A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.

Abstract

We have used phospho-specific antibodies to re-examine the multisite phosphorylation of c-Jun in murine RAW macrophages and embryonic fibroblasts. Our results indicate that JNK isoforms are required and sufficient for the phosphorylation of Thr91 and Thr93, as well as the phosphorylation of Ser63 and Ser73, in response to LPS or anisomycin in macrophages and TNFalpha or anisomycin in fibroblasts. However, the phorbol ester (TPA) and EGF-induced phosphorylation of Ser63 and Ser73 is mediated by ERK1/ERK2, as well as JNK1/JNK2, in fibroblasts from wild-type mice and by ERK1/ERK2 alone in fibroblasts from JNK-deficient mice. The phosphorylation of Thr239 is catalysed by GSK3 and the phosphorylation of Ser243 by an as yet unidentified protein kinase. The inhibition of GSK3 is not required for the dephosphorylation of Thr239 in response to LPS, and nor is the phosphorylation of Thr91 and Thr93 required for the TPA- or EGF-induced dephosphorylation of Thr239 in fibroblasts. The agonist-induced dephosphorylation of Thr239 may involve a conformational change that exposes Thr239 to dephosphorylation and/or the activation of a Thr239 phosphatase.

Fig. 1. Characterization of phospho-specific antibodies that recognize particular phosphorylation sites on c-Jun. Bacterially expressed c-Jun was left unphosphorylated (no kinase, NK) or maximally phosphorylated with either JNK, GSK3β or ERK2, and 100 ng aliquots spotted on to nitrocellulose membranes and immunoblotted using the phospho-specific antibodies indicated, or an antibody that recognizes phosphorylated and unphosphorylated c-Jun equally well, in the presence or absence (none) of the competitor peptides shown. The sequences of these peptides are given in Materials and methods. The prefix ‘p’ denotes the phosphorylated residue. (A) Examination of the specificity of the antibodies that recognize c-Jun phosphorylated at Ser63 or Ser73. (B) Specificity of the antibodies that recognize c-Jun phosphorylated at Thr91 or Thr93. (C and D) Specificity of the antibodies that recognize c-Jun phosphorylated at Thr239 or Ser243.

Fig. 2. Effect of LPS and anisomycin on the phosphorylation of six sites on c-Jun in RAW macrophages. Macrophages were stimulated with LPS (100 ng/ml) or anisomycin (10 µg/ml) for the times indicated and lysed. c-Jun was immunoprecipitated from the lysates denatured in SDS, subjected to SDS–PAGE and transferred to nitrocellulose. The membranes were immunoblotted with antibodies that recognize each of the six phosphorylation sites on c-Jun, as well as with an antibody that recognizes the phosphorylated and unphosphorylated forms of c-Jun equally well (A and B). Further aliquots of the lysates were immunoblotted with antibodies that recognize the active phosphorylated forms of several MAP kinases, namely JNK isoforms, p38α and ERK1 and ERK2, as well as with antibodies that recognize the phosphorylated and dephosphorylated forms of these kinases equally well (C and D). The prefix ‘p’ denotes the phosphorylated residue. Similar results were obtained in several independent experiments.

Fig. 3. Inhibitors of GSK3 induce the dephosphorylation of c-Jun at Thr239 without affecting the phosphorylation of Ser243. RAW cells were incubated for 1 h with or without LiCl (10 mM) or Kenpaullone (20 µM) and lysed. c-Jun was immunoprecipitated from the lysates, denatured in SDS and immunoblotted using antibodies that recognize c-Jun phosphorylated at Thr239 or Ser243 or with an antibody that recognizes the phosphorylated and unphosphorylated forms of c-Jun equally well (see legend to Figure ). The prefix ‘p’ denotes the phosphorylated residue. Similar results were obtained in several independent experiments.

Fig. 4. The inhibition of GSK3 is not required for the LPS-induced phosphorylation of c-Jun. RAW cells were stimulated for 30 min with 100 ng/ml LPS (A and B) or 10 µg/ml anisomycin (C) and lysed. (A and C) Lysates were subjected to SDS–PAGE and immunoblotted using antibodies that recognize GSK3 phosphorylated at Ser21 (GSK3α) and Ser9 (GSK3β), or GSK3 phosphorylated at Tyr279 (GSK3α) and Tyr216 (GSK3β) or with an antibody that recognizes the unphosphorylated and phosphorylated forms of GSK3β equally well. (B) The cells were incubated with or without 2 µM PD 184352 and/or 100 nM wortmannin prior to stimulation for 30 min with 100 ng/ml LPS and then lysed. c-Jun was immunoprecipitated from the lysates, denatured in SDS and immunoblotted using antibodies that recognize c-Jun phosphorylated at Thr239 or Ser243. The lysates were also immunoblotted with the anti-GSK3 antibodies in (A and C), as well as with antibodies that recognize the phosphorylated forms of ERK1 and ERK2 or PKB phosphorylated at Ser473. The prefix ‘p’ denotes the phosphorylated residue. Similar results were obtained in several independent experiments.

Fig. 5. SB 203580 and PD 184352 do not prevent the LPS- or anisomycin-induced phosphorylation of c-Jun at Ser63, Ser73, Thr91 or Thr93. RAW cells were stimulated for 30 min with 10 µg/ml anisomycin (A) or 100 ng/ml LPS (B) and lysed. c-Jun was immunoprecipitated from the lysates, denatured in SDS and immunoblotted using antibodies that recognize c-Jun phosphorylated at Ser63, Ser73, Thr91 or Thr93 as described in the legend to Figure . Further aliquots of the cell lysates were immunoblotted (without immunoprecipitation) using antibodies that recognize the active phosphorylated forms of ERK1 and ERK2, and MAPKAP-K2 phosphorylated at Thr334. The prefix ‘p’ denotes the phosphorylated residue. Similar results were obtained in several independent experiments.

Fig. 6. Phosphorylation of the four N-terminal sites in c-Jun in WT and JNK-deficient fibroblasts. Fibroblasts from WT (WT) and JNK-deficient (JNK–/–) mice were stimulated (A) for 30 min with anisomycin (10 µg/ml), (B) for 15 min with TNFα (10 ng/ml), (C) for 30 min with TPA (100 ng/ml) and (D) for 15 min with EGF (100 ng/ml). Following cell lysis, c-Jun was immunoprecipitated from the lysates, denatured in SDS, subjected to SDS–PAGE and transferred to nitrocellulose. The membranes were immunoblotted with antibodies that recognize c-Jun phosphorylated at Ser63, Ser73, Thr91 and Thr93, as well as with an antibody that recognizes the phosphorylated and unphosphorylated forms of c-Jun equally well. The prefix ‘p’ denotes the phosphorylated residue. Similar results were obtained in several independent experiments.

Fig. 7. Effect of PD 184352 on the TPA-induced phosphorylation of the c-Jun in WT and JNK-deficient fibroblasts. Fibroblasts from WT (WT) (A and C) and JNK-deficient (JNK–/–) (B and D) mice were incubated for 1 h with or without 2 µM PD 184352 and then stimulated for 30 min with TPA (100 ng/ml). The cells were lysed and immunoblotting carried out as described in the legend to Figure . Similar results were obtained in several independent experiments. (E) Bacterially expressed c-Jun was left unphosphorylated (no kinase, NK) or phosphorylated with ERK2 and aliquots spotted onto a nitrocellulose membrane. They were then immunoblotted using the phospho-specific antibodies indicated or an antibody that recognizes phosphorylated and unphosphorylated c-Jun equally well. The prefix ‘p’ denotes the phosphorylated residue. Similar results were obtained in several independent experiments.